Aminoacyl tRNA synthetases establish the rules of the genetic code by catalyzing the aminoacylation of transfer RNAs. The chemical invariance of the twenty amino acid building blocks of proteins is well established. The only known extensions to this invariant set are formyl-methionine (1) and selenocysteine (2), both incorporated in response to punctuation signals during translation in certain organisms.
Thus, although species have colonized dissimilar terrestrial habitats throughout geological times, this diversification has not been mirrored in the evolution of organisms to include specialized sets of amino acids. For instance, thermophilic, mesophilic, and psychrophilic organisms all assemble proteins by combining the same types of twenty canonical amino acids into different protein sequences. Standing as the “missing link” between alanine and valine (3), aminobutyrate (Abu, also known as butyrine) can be generated by transamination from the physiological metabolite 2-oxo-butyrate and should thus be considered as a latent metabolite (4). Its absence is therefore particularly conspicuous in the proteins of extant organisms.
The selection of amino acids for protein synthesis is done by aminoacyl tRNA synthetases. Typically, each of twenty synthetases catalyzes the attachment of its cognate amino acid to the 3′-end of its cognate tRNA and amino acids are, in this way, associated with specific triplets of the genetic code (5). The active site of several of these enzymes inherently lack the capacity to discriminate between closely similar amino acids at a level sufficient to explain the high accuracy of the code. For that reason, a given enzyme may misactivate closely similar (in size and shape) amino acids at a low frequency (0.1 to 1%) (6). To correct these errors, in many cases, a hydrolytic editing function, at a separate active site, has developed (7-10). One example of a synthetase that has editing activity is valyl-tRNA synthetase (ValRS), which misactivates the isosteric natural amino acid Thr (9), as well as the non-natural Abu (11). Misactivation of these amino acids leads to transient mischarging of tRNAVal, followed by hydrolytic deacylation (editing) of the mischarged amino acid from the tRNA.
The present work aimed to establish conditions of artificial selection that promoted usage of non-canonical amino acids, such as Abu, that were not retained by natural selection. Others have attempted to incorporate a non-canonical amino acid into a protein by introducing a foreign, “orthogonal” tRNA/synthetase pair that can insert the amino acid at a specialized stop codon (12).
However, such approaches are laborious, as they require selection, identification, cloning, and study of individual mutant strains.
In order to facilitate the in vivo production of proteins comprising noncanonical amino acids, it would be desirable to have a rapid and generalized method allowing to genetically modify and select cells capable of achieving the in vivo production of such proteins.
Such a desirable method will allow to enlarge the chemistry of translation by having a non-canonical amino acid “infiltrate” all of the codons normally associated with one of the natural amino acids. Indeed, by assigning two amino acids (a cognate and a non-cognate) to a specific set of codons so as to provide a selective advantage to the reprogrammed cells, global changes in the amino acid compositions of all cellular proteins could be made. The present invention addresses this need.